Project Summary/Abstract Traumatic brain injury (TBI) is a leading cause of death and disability with approximately 1.7 million incidents annually at a total cost of over $76 billion in 2000 according to the Center for Disease Control and Prevention. There is an urgent need to develop new treatments that will limit secondary injury associated with inflammation, decrease blood-brain barrier permeability, increase neurogenesis, and overall improve clinical outcome in TBI. To that end, cell therapies using mesenchymal stem cells (MSC) and MSC-like cells have demonstrated some efficacy in pre-clinical models and similar indications. There are a number of technical challenges in using cell therapies to treat traumatic injuries, such as their rapid clearance from circulation and a limited ability to home to sites of injury and the brain. Our team has developed a set of bioengineering tools to precisely control the expression of multiple genes integrated into a landing pad. Here we propose to combine this new technology with our ongoing studies of MSC as a promising therapeutic for TBI. Specifically we propose to express a combination of an important damage homing protein (CXCR4) and a leukocyte adhesion molecule (PSGL-1) along with an enzyme that increases PSGL-1 binding affinity to markers of injury and inflammation (FUT7/SLeX). We will test different levels of expression in combination to select populations of MSC that are specified to detect damaged endothelia in vitro and then in vivo in order to increase the localization of MSC to the CNS following TBI. We hypothesize that by increasing the homing and attachment of MSC to injured tissue, we can increase the efficiency of several key mechanisms of action used by MSC to improve TBI outcomes, such as local paracrine support and modulation of immune cell activity. This study includes a proof-of-concept experiment utilizing an animal model of TBI, where modified MSC are tested for the ability to improve both short-term biomarkers of injury and long-term behavioral outcome measures of memory and cognition. The successful completion of this study will demonstrate that we can specifically target MSC to inflamed endothelia, increase the potency of MSC to treat TBI, and prove a novel bioengineering approach can significantly advance the translational applications of cell therapies.